The intrinsic advantage of biological processing is that bacteria are able to convert, at low temperature, a large array of compounds with different chemical features to few or single product(s) which can be obtained in high purity form. Anaerobic fermentation/digestion is a complex biochemical process of biologically mediated reactions by a consortium of microorganisms to convert organic compounds into methane and carbon dioxide, as final products. It typically involves four stages: hydrolysis – chemical reaction that involves solubilisation of particulates and breakdown of large polymers into simple monomers; acidogenesis – biological reaction where simple monomers are converted into VFAs; acetogenesis – biological reaction where VFAs are converted into acetic acid, carbon dioxide and hydrogen; methanogenesis – the final biological stage where acetates are converted into methane and carbon dioxide, while hydrogen is consumed.
PAPER BIO-OIL FERMENTATION
Fermentative pathways are able to partially deoxygenate sugars with high selectivity, in comparison to chemical pathways [104]. On the other hand, pyrolysis oil contains several compounds at percent level which are toxic to microorganism, hence process optimisation is important to exploit biological transformation, overcoming the toxic effect of pyrolysis derived chemicals [74].
A UASB reactor (33mL working volume) was inoculated with mixed bacterial sludge and acclimatised to the bio-oil feed for a period of 61 days. The sludge was retained in the reactor throughout the fermentation period, while the bio-oil had a HRT of 2 days.
Fig. 3.6 shows the trend in the conversion of paper bio-oil substrate into VFA intermediate and
triethylcitrate (TEC). Data reported are the mean value of duplicate, which showed an acceptable relative standard deviation not more than 21% and were explicated as gCODL-1 in
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order to compare directly the various products in term of chemical energy. The first 18 days of acidogenesis produced exclusively VFAs, mainly acetic and propionic acids, with as high as 1.6 gCODL-1 VFA (Fig. 3.6). Subsequently, VFA production gradually decreased to zero, while the formation of ethylcitrates (EC: TEC and DEC) was observed from the 19th day until the end of the fermentation process (Fig. 3.7). A sharp increase in ethylcitrates content was observed at the start of the citrate production with a peak value of 2.8 ± 0.1 gCODL-1, after which it gradually decreased to peak values of roughly 1.0 ± 0.2 gCODL-1.
Fig. 3.6: Trend of VFA and triethylcitrate generated during acidogenic fermentation of paper
bio-oil 0.00 0.50 1.00 1.50 2.00 2.50 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 gC OD L -1 Time (days)
Acetic acid Propionic acid Isobutyric acid
Butyric acid Isovaleric acid Valeric acid
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Fig. 3.7: Trend of DEC, TEC, total citrate (DEC and TEC) generated during acidogenic
fermentation of paper bio-oil
Looking at the product yield (Fig. 3.6 and 3.7), with an inlet COD of 5.7g L-1, the maximum VFA peak produced corresponded to 28% while that of EC corresponded to to 49 ± 2 %. The former is comparable with the VFA production result reported by Torri et al. who used corn stalk pellets pyrolysis oil as substrate for VFA intermediate production [74]. In totality, TEC were in much higher yield than DEC, with at least two third of the EC produced being TEC (Fig. 3.7). It is also worth mentioning that the undulating trend of VFA and EC produced by the mixed culture is consistent with literature [74] and is partly a function of the response of bacteria to use carbon substrate and convert per time.
The acidogenic fermentation which lasted 61 days was sucessful in the conversion of anhydrosugars in the bio-oil (mainly levoglucosan, see Table 3.1) to several fermentation products. The MMC was able to convert the pyrolysis products into among several products, VFAs, our original compound of interest, and ethylcitrates – first time this is reported as a product of acidogenic fermentation. While VFAs have been considered the best substrates for
0.00 0.50 1.00 1.50 2.00 2.50 3.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 gCOD L -1 Time (days)
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PHA accumulation by MMC [73], it was thought interesting to investigate if our MMC can also accumulate PHA from TEC (a triester of ethanol and citric acid, and the major fermentation product), to know the possibility of using directly the fermentation products for PHA accumulation. Hence, the reason for the three SBRs selection in section 2.2.6 (R1 – TEC as substrate, R2 – VFA as substrate, R3 – Ethanol as substrate).
MICROALGAE AQUEOUS PHASE ACIDOGENESIS
A UASB reactor (33mL working volume) was inoculated with mixed bacterial sludge and acclimatised to the microalgae HTL-AP feed for a period of 64 days. The sludge was retained in the reactor throughout the fermentation period, while the substrate had a HRT of 2 days.
Fig. 3.8 and 3.9 show the trend in the conversion of microalgae HTL-AP substrate into VFA
intermediate and EC. The presence of VFAs can be observed at the beginning of the fermentation (day zero, 0.5gCODL-1 ) and gradually decreased until the 22nd day to 0.04gCODL-1, after which it increased with a peak value of 1.0gCODL-1. Meanwhile, the period of low VFA production afforded EC yield, with a maximum yield of 0.7gCODL-1 on the 22nd day, and then decreased gradually to 0.05gCODL-1.
Fig. 3.8: Trend of DEC, TEC, total citrate (DEC and TEC) generated during acidogenic
fermentation of microalgae HTL-AP
0.000 0.200 0.400 0.600 0.800 1.000 1.200 0 10 20 30 40 50 60 70 gC OD L- 1 Time (days)
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Fig. 3.9: Trend of VFA and triethylcitrate generated during acidogenic fermentation of
microalgae HTL-AP
Form the product yield in Fig. 3.8 and 3.9, an inlet substrate COD of 13.1g L-1 afforded peak values that correspond to 7.6% VFA and 5.3% EC yields. DEC was in a higher yield than TEC and contributed to more than two third of the total citrates produced. This is hypothesized to be a result of selective inhibitors in the metabolic pathway that hampers the further esterification to DEC. The presence of VFAs observed at the beginning of the fermentation is quite in accordance with the report of Zhou et al. [84] on the presence of some VFAs such as acetic acid in the AP of some algae species. Furthermore, the low product yield observed was due to the formation of ammonia (formed as a result of the high nitrogen content in the substrate), which inhibited the acidogenic process, and in turn, evolved methane. Therefore, it can be hypothesized that microalgae AP could act as a strong inhibitor for acidogens, consequently leading to poor acidogenic fermentation products yield.
By juxtaposing the paper oil and microalgae AP fermentation processes, the following were observed: First, at each point of analysis, the production of VFA is inversely proportional to EC production, during the microalgae AP fermentation process (Fig. 3.8). Second, unlike paper oil acidogenesis, DEC produced with microalgae HTL-AP fermentation were in a higher yield
0.000 0.200 0.400 0.600 0.800 1.000 1.200 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 gC OD L -1 Time (days)
Acetic acid Propionic acid Isobutyric acid butyric acid
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than TEC. Thirdly, the microalgae AP acidigenesis produced a wider variety of VFAs though in lower concentration when compared to paper pyrolysis oil fermentation.